Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain.

Background Bovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The phylogenetic relationships between two isolates of Theileria annulata, and 36 Theileria spp., as well as 6 outgroup including Babesia spp. and coccidian protozoa were analyzed using the 18S rRNA gene sequence. Methods The target DNA segment was amplified by PCR. The PCR product was used for direct sequencing. The length of the 18S rRNA gene of all Theileria spp. involved in this study was around 1,400 bp. Results A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iran and Iraq isolates, and other species of Theileria available in GenBank. In the constructed tree, Theileria annulata (Iran vaccine strain) was closely related to other T. annulata from Europe, Asia, as well as T. lestoquardi, T. parva and T. taurotragi all in one clade. Conclusion Phylogenetic analyses based on small subunit ribosomal RNA gene suggested that the percent identity of the sequence of Iran vaccine strain was completely the same as Iraq sequence (100% identical), but the similarity of Iran vaccine strain with other T. annulata reported from China, Spain and Italy determined the 97.9 to 99.9% identity.


Introduction
ovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The two most important species in cattle and water buffalo are T. parva, as the agent for causes east coast fever, and T. annulata, which causes tropical theileriosis. Theileria parva occurs in 13 countries in sub-Saharan Africa causing East Coast fever (ECF), Corridor disease, and January disease. Theileria annulata, the cause of tropical theileriosis, occurs in large parts of the Mediterranean coast of north Africa, extending to northern Sudan, and southern Europe. Southeastern Europe, the near and Middle East, India, China and central Asia are also affected (1)(2)(3). Theileria is classified based on microscopic observations, ultrastructural features, lifecycle, geographic region and vertebrate and nonvertebrate host (4). Clinical classification categorizes Theileria to malignant, moderate and benign species. Nonetheless, the exact taxonomic Theileria spp. have been difficult to establish and the subject of considerable debate (5)(6)(7). There is a number of factors makes complexity of assigning taxonomic positions, including similar morphology among this group of parasites regardless of vertebrate host, incomplete lifecycle data, serologic tests that are not specific enough to discriminate individual species in the presence of mixed infections, and the difficulty of obtaining pure isolates for studies when the circulating parasitemia may be very low (8)(9)(10). The advances in molecular biology and specially sequence data analysis allowed the researchers to identify and characterize the hemoparasites species in particular Theileria group. Ribosomal RNA is the most abundant constituent of nucleic acids in any non-viral organism with the eukaryotic RNA transcription unit consisting of the large and small subunit (18S rRNA) and the 5.8S rRNA gene (11). The 18S rRNA gene is increasingly accepted as a widely used marker for characterization, taxonomic classification, and phylogenetic analysis and this gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (6,10,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). The conserved function and structure of the 18S rRNA molecule allow sequences to be aligned, even among divergent species. However, the molecule also possesses phylogenetically informative variable regions that are useful for determining relationships among species (23). In this study, we decided to determine the phylogenetic position of T. annulata infected cell line S15 Iran vaccine strain and T. annulata Iraq field isolate with 36 different Theileria spp. and six out-group protozoan parasites using 18S rRNA gene sequences comparison.

Parasites
Two T. annulata strain/isolates were used in this study. Theileria annulata infected cell line (S15 Iran vaccine strain, Tehran, Vasfenard) and Iraq T.annulata field isolate were provided from Protozoology and Vaccine Production Department of Razi Institute and Duhok Province, Kurdistan of Iraq respectively.

DNA isolation
Proteinase K and further phenol chloroform purification were performed for DNA extraction (24). Briefly, after treating the cells with lyses buffer, followed by centrifugation, proteinase K and SDS solution was added to the pellet, and then was incubated until most of the cellular protein was degraded. The digest was deproteinized by phenol/chloroform/isoamyl alcohol extraction, recovered by ethanol precipitation, then was dried and resolved in TE buffer. DNA concentration was determined either by agarose gel electrophoresis and spectrophotometry (A260) and measuring the ratio of A 260 /A 280 . Moreover, quality of the isolated DNA was evaluated by agarose gel electrophoresis.

PCR Primer Design
The specific primers were designed based on T. annulata 18S ribosomal RNA gene sequence (accession # EU083801) (by CinnaGen, Iran). Two primer pairs were designed in order to span the major hyper variable regions along the 18S ribosomal RNA gene sequence (Gene Runner program, Version 3.05). The first two primers, F1 (5' GGC GGC GTT TAT TAG ACC 3') and R1 (5' TCA ATT CCT TTA AGT TTC AGC C 3') were used to amplify bases between 186-1093 and the second primers, F2 (5'CAG ATA CCG TCG TAG TCC 3') and R2 (5' CCT TGT TAC GAC TTC TCC 3') were applied to amplify bases between 945-1714 of T. annulata 18S ribosomal RNA gene sequence (EU083801) and these two primers sets covered the majority length of 18S ribosomal RNA gene sequence with 127 bp overlapping.

PCR product detection and sequencing
Amplified PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized by UV transillumination. PCR products were cleaned and extracted from agarose gel and were submitted for bidirectional DNA sequencing by using chain termination method (MWG, Ger-many). The provided sequences from F1R1 and F2R2, first were merged and offered to be aligned for multiple sequence alignment and phylogenetic study of Theileria spp. and outgroups as well.

Sequence alignment and phylogenetic analysis
The DNA sequences of 18S rRNA gene obtained from two studied T. annulata samples and 42 sequences of 18S rRNA gene sequences including 36 Theileria spp., 4 Babesia spp. and two coccidian protozoa were accessed from GenBank. The sequences were aligned by Clustal W multiple alignments program (25). The alignment was manually edited in BioEdit and truncated to the size of the smallest sequence (~1,400bp). Phylogenetic tree was constructed by using DNADist Neighbor-Joining method (version 3.6a2.1), sequence identity matrix of all sequences were computed as well (BioEdit phylogeny package, Version 7.0.1).

Nucleotide sequence accession numbers
The 18S rRNA gene sequences of the T. annulata S15 Iran vaccine strain and T. annulata field isolate from Iraq, Duhok have been submitted to GenBank and can be retrieved under accession numbers of HM628581, and HM628582 respectively. All 42 small subunit ribosomal RNA gene sequences were used for this phylogenetic study were listed in Table 1.

Results
The expected amplicons with sizes of 770 and 908 base pairs (bp) were observed in all of the examined samples (Fig. 1). The sequences of the 18S rRNA gene of the T. annulata Iran vaccine strain and Iraq (Duhok isolate) were determined from the overlapping flanking sequences of two generated PCR fragments. The sequencing of the PCR products yielded 1424 and 1413 base pair length for two Iran and Iraq samples respectively. The sequences were then subjected to phylogenetic analysis by using the BioEdit programme. The sequence identity matrix was also determined and showed 88.2-100 % homology between T. annulata 18S rRNA sequence of S15 Iran vaccine strain and Theileria spp. from all over the world (

Entropy plot
The alignment was manually edited in BioEdit software and truncated to the size of the smallest sequence (1,413 bp). After resizing the aligned 44 sequences, in order to have the correct comparison, the designed entropy plot was plotted and this design showed the amplified 1413 bp length of the 18S rRNA gene sequence in this study, spans most of the hypervariable regions exist along the alignment (Fig. 2).

Sequence alignment and phylogenetic analysis
The phylogenetic tree was constructed based on the T. annulata Iran S15 vaccine strain, Iraq isolate, 36 Theileria spp. and 6 out-group sequences including Babesia spp., Toxoplasma gondii and Isospora suis sequences (Table 1). Theileria sequences was divided into six clades in the constructed tree, including "Q" clade; consists T. equi, "Y" clade; includes T. youngi and T. bicornis, "O" clade; contains T. ovis, T. capreoli and T. velifera, "M" clade; includes T. mutans, "B" clade; consists T. buffeli, T. sergenti, T. sinensis, T. separata and T. orientalis, "A" clade; includes T. annulata, T. parva, T. lestoquardi and T. taurotragi, and two more clades for outgroup sequences; "Babesia" clade; consists B. ovis, B. divergens, B. motasi and B. cabali, and "Coccidian" clade; contains Toxoplasma gondii and Isospora suis (Table 1 and Fig. 3). There are eight T. annulata 18S rRNA gene sequences in clade "A", identity percent of T. annulata Iran vaccine strain and other T. annulata in this clade are 99.2% to 100%.  The identity percent between T. annulata Iran strain and T. lestoquardi was 99.5% and this similarity rate was more than the identity percent between T. annulata Iran and Italy or China, Xinjiang isolates.

Discussion
Based on available literature, this is probably the first phylogenetic analysis, molecular characterization of T. annulata infected cell line of Iran vaccine strain, and a field isolate from clinically infected cattle in Iraq, Duhok by using 18S rRNA gene sequence. One of the most important points in Good Manufacturing Practice (GMP) is to characterize the local vaccine seed in Razi Vaccine and Serum Research Institute, Karaj, Iran. Therefore, we decided to classify the T. annulata Iran vaccine strain cell line. Hence, we focused on to establish the phylogenetic relationships of Iran T. annulata vaccine strain with other Theileria species using 18S rRNA gene sequences.
Basically, there are three steps in all phylogenetic analysis; multiple alignment of the sequences, distance calculation and tree construction. Using taxa (the outgroup) that are known to fall outside of the group of interest (the ingroup) is the way to root tree. In this study, we applied Babesia species and two coccidian parasites to root Theileria species. In addition, Theileria species are host and vector specific (26,27) but in some countries more than one species can infect animals, which causes a problem in diagnosis and epidemiology. Although recent molecular studies suggest that the genus Theileria including T.
annulata, T. parva and T. lestoquardi are very similar in features of microscopic characteristics; these species are phylogenetically distinct and can be differentiated by accurate molecular techniques (28,29). Although, Theileria spp. are tick-transmitted and the parasite is fully corresponded to the specific hosts, and this association makes a geographical distribution of the Theileria species around the world, but the phylogenetic analysis clearly shows close relationship of different species, T. annulata, T. lestoquardi, T. parva and T. taurotragi in constructed tree all are within clade "A" (Fig. 4).  In conclusion, according to the data presented here, there are high homologies between T. annulata Iran 18S rRNA gene sequence strain with other T. annulata from above mentioned countries, in particular Iraq and Turkey. Therefore, if this phylogenetic data correlate with immunological response of susceptible cattle, it might be a new sight to find an efficient vaccine to control and prevention of Tropical Theileriosis through molecular epidemiological methods.